Method And Device For 3D Reconstruction Of The Distribution Of Fluorescent Elements

ABSTRACT

A device and method for processing fluorescence signals emitted after excitation by radiation coming from a radiation source, by at least one fluorophore with a lifetime τ in a surrounding medium, which signals are detected by detection means, and which method includes the calculation, on the basis of detected fluorescence signals, of values of a variable, independent of τ, of the position or the distribution of fluorophore in said medium.

CROSS-REFERENCE TO RELATED PATENT APPLICATION OR PRIORITY CLAIM

This application is a continuation of co-pending U.S. application Ser.No. 11/888,579, filed Jul. 31, 2007 which claims the benefit of a FrenchPatent Application No. 06-07065, filed on Aug. 2, 2006, in the FrenchIntellectual Property Office.

TECHNICAL FIELD AND PRIOR ART

This invention relates to the field of fluorescence molecular imaging onbiological tissue by time-resolved optical methods.

It applies in particular to optical molecular imaging on small animalsand optical molecular imaging on humans (brain, breast, and other organswhere fluorophores can be injected).

The optical fluorescence molecular imaging optical techniques are nowbeing increasingly developed owing to the use of specific fluorescentmarkers. These bind preferentially to the target cells of interest (forexample cancer cells) and provided better detection contrast thannon-specific markers. These techniques are designed to spatially locatethe fluorescent markers, but also to determine the concentrationthereof.

Optical tomography systems use various light sources. There aretherefore continuous-mode apparatuses, frequency-mode apparatuses (thatuse frequency-modulated lasers) and finally apparatuses operating intemporal mode, which use pulsed lasers.

The time data is the data that contains the most data content on thetraversed tissue, but for which the reconstruction techniques are themost complex. The measurement at each acquisition point is indeed atime-dependent function (called TPSF for “Temporal Point SpreadFunction”).

It is sought to extract simple parameters of the TPSF, of which thetheoretical expression is known. Then, the resolution of the reverseproblem makes it possible to find the distribution of the fluorescentmarkers.

In document WO 2006/032151, the first three moments of the time curves,which are called “normalised moments” because they are the moments ofthe curves normalised by the excitation curve, are extracted.

Now, the moments of the time curves are dependent on the knowledge ofthe fluorescence lifetime. Consequently, in this document, to solve thereverse problem, the fluorescence lifetime is assumed to be known.However, this lifetime can be sensitive to the environment and isdifficult to measure in vivo. A poor choice of this parameter leads toerroneous results.

The article of A. T. N. Kumar et al. “Fluorescence lifetime-basedtomography for turbid media,” Opt. Lett. 30(24), 3347-3349 (2005)describes a method that reconstructs one or more lifetimes. In fact, itis based on the determination of different lifetimes on the decaycurves; then on a reconstruction based on their respective amplitudes.An approximation on the lifetime is made at the outset. This techniqueis particularly advantageous if there are two clearly distinctlifetimes.

The article “Time-Domain Fluorescence Molecular Tomography Based onGeneralized Pulse Spectrum Technique”, Fen Gao, Wei Liu et al.,Proceedings BIOMED 2006 describes a method based on the Laplacetransforms of the time curves at two frequencies, which requires tworeconstructions, one on the concentration and the other on the lifetime.This method is more complex and costly in calculation time because it isbased on numerical calculations to obtain the Laplace transforms (theanalytical expression is not easy to find).

Therefore, there is the problem of determining the spatial distributionof fluorophores in a diffusing medium, without prior knowledge of thefluorescence lifetime.

DESCRIPTION OF THE INVENTION

The invention first relates to a method for processing emittedfluorescence signals, after excitation by radiation from a radiationsource, by at least one fluorophore with a lifetime τ in a surroundingmedium, which signals are detected by detection means, and which methodcomprises:

-   -   the detection of a plurality of fluorescence signals (Φ_(fluo))        emitted by the fluorophore(s) in the surrounding medium, each        signal corresponding to a relative position, on the one hand of        the fluorophore(s) and on the other hand of the source and the        detection means,    -   the calculation, based on these detected fluorescence signals,        of values of a variable, independent of τ but dependent on the        position or the spatial distribution of fluorophores in said        medium,    -   the determination of the position or the spatial distribution of        fluorophores in said medium on the basis of the values of said        variable.

The fluorophore(s) can be bonded to biological tissue, for exampletarget cells of interest (for example cancer cells).

The detection of the signal(s) is preferably performed in a measurementwindow making it possible to recover almost all of the photons emittedby the fluorescence. The calculation of the values of a variableindependent of the lifetime can therefore be done by taking into accountthe entirety of the signal(s) detected in this measurement window.

A method according to the invention can, for example, be performedwithout the fluorescence lifetime by considering a normalisedmeasurement, for example to the measurement that gives the shortestfluorescence lifetime.

The variable independent of τ can also result from a normalisedfrequency function, for example with respect to a specific fluorescencesignal.

This method can be adapted to transformations other than the mean time(for example, the Mellin-Laplace transforms), which make it possible todo without the lifetime owing to the normalisation.

According to an embodiment, said variable independent of τ results fromthe difference between the mean time τ calculated for each fluorescencesignal and the mean time calculated for a specific fluorescence signal,preferably that for which the calculation time is minimal.

According to another embodiment, the independent variable results fromthe Mellin-Laplace transforms of the fluorescence signals.

The determination of the position or the spatial distribution offluorophores in the medium can be achieved by a method of reversal usingvalues of the variable. The reversal method can result from theminimisation of an error function between the measurement and saidvariable, for example using a simplex method.

According to another embodiment, the determination of the spatialdistribution of fluorophore in said medium implements the resolution ofa system of linear equations:

M=P×C,

-   -   where M is a measurement column vector, P is a weight matrix and        C is a distribution column vector.

According to the invention, it is possible to calculate the first momentof the curve of the fluorescence signal as a function of time.

A method according to the invention can also comprise a preliminary stepof measuring the fluorescence signal emitted by the surrounding medium,in the absence of fluorophore, then a step of correcting thefluorescence signals (Φ_(fluo)) emitted by the fluorophore in itssurrounding medium.

The invention also relates to a device for processing fluorescencesignals emitted by at least one fluorophore, with a lifetime τ in asurrounding medium, comprising:

-   -   a source of radiation for excitation of said fluorophore,    -   detection means for detecting a fluorescence signal emitted by        said fluorophore,    -   means for performing a relative movement of the source and the        detection means with respect to the fluorophore,    -   means for calculating values of a variable independent of τ, on        the basis of a plurality of fluorescence signals (Φ_(fluo))        emitted by the fluorophore into its surrounding medium, with        each signal corresponding to a relative position, on the one        hand of the fluorophore and on the other hand of the source and        the detection means,    -   means for determining the position (in the case of a single        fluorophore) or the spatial distribution (in the case of a        plurality of fluorophores) of fluorophore(s) in said medium on        the basis of the values of said variable.

The detection means are, for example, of the TCSPC type or camera-typemeans.

A device according to the invention can also comprise means for visualor graphic representation of the position or the spatial distribution ofthe fluorophore(s).

The variable independent of τ can result from a normalised frequencyfunction, with respect to a specific fluorescence signal.

According to an embodiment, the variable that is independent of τresults from the difference between the mean time τ calculated for eachfluorescence signal and the mean time calculated for a specificfluorescence signal.

This specific fluorescence signal is advantageously the one for whichthe mean time calculated is minimal.

According to an embodiment, the means for determining the position orthe spatial distribution of fluorophores in the medium implement aminimisation of an error function of the values of said variable, whichminimisation can be a processing operation by adjustment using a simplexmethod.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B each show an example of an experimental device forimplementing the invention.

FIGS. 2A and 2B respectively show a series of laser pulses and singlephotons emitted, and a fluorescence curve obtained on the basis of datarelating to the single photons.

FIG. 3 shows examples of fluorescence time decay curves, with andwithout fluorophore.

FIG. 4 diagrammatically shows the relative position of a source-detectorpair and the inclusion of s.

FIG. 5 shows fluorescence data for various positions of a fluorophoreunder excitation and detection fibres.

FIG. 6 shows mean times calculated on the basis of fluorescencemeasurements for various positions or depths of a fluorophore.

FIG. 7 shows three fluorescence time decay curves, one resulting from ameasurement and two from simulations.

FIG. 8 shows a grid of detectors.

FIG. 9 shows the change in detection mean times for various positions ofthe detector-fluorophore assembly.

FIG. 10 shows the change in detection mean times as a function of thetotal optical path covered by the photons.

DETAILED DESCRIPTION OF SPECIFIC EMBODIMENTS

FIG. 1A is an example of an experimental system 2 using, as a detector4, a photomultiplier and a TCSPC (Time Correlated Single PhotonCounting) card, actually integrated in an assembly of data processingmeans 24.

The light is transmitted by a pulsed radiation source 8, sent andcollected by fibres 10, 12, which can be moved. The two fibres can bemounted on means for vertical and horizontal translation movement(according to the X and Y axes of FIG. 1). The distance d between thefibres is, for example, around 0.2 cm.

The radiation pulse source 8 can also be used as means for activatingthe TCSPC card (see the link 9 between the source 8 and the means 24).

According to a specific embodiment, the source 8 is a pulsed laserdiode, with a wavelength of 631 nm and a repetition rate of 50 MHz.

The laser light preferably passes through an interference filter 14 soas to remove any light with a wavelength higher than the excitationwavelength.

The fibre 12 collects the light coming from the medium 20 studied. Aninterference filter 16 and a coloured filter absorbing the highwavelengths can be placed in front of the detector 4 so as to select thefluorescent light (for example: λ>650 nm, with the source being at thewavelength 631 nm) of a fluorophore 22 placed in the medium 20 and tooptimise the elimination of the excitation light.

According to the TCSPC technique (for “Time Correlated Single PhotonCounting”), using a photomultiplier, a photon emitted by the fluorophoreafter a radiation source pulse, is detected.

The system therefore enables time-resolved detection of fluorescencepulses. It makes it possible to recover almost all of the fluorescencephotons.

FIG. 2A shows a series of laser pulses

Li (i=1-4) and a series of corresponding single photons pi (i=1-4). Eachphoton is in fact detected with respect to the start of thecorresponding pulse: in FIG. 2A, ti represents the time lapsed betweeneach laser pulse Li and the time of detection of each photon pi.

It is then possible therefore to establish a statistical distribution,as shown in FIG. 2B, of the number of fluorescence photons detected, asa function of the time lapsed t with respect to each laser pulse. Such acurve Φ_(fluo) (t), which, as seen (also in FIGS. 3, 5 and 7), makes itpossible to use all of the data in a large time window, on each side ofthe maximum intensity point (and not only in the rising portion of thesignal) can then be processed so as to obtain the characteristic data aswill be explained below.

Electronic means 24 such as a microcomputer are programmed to store andprocess the data of the TCSPC card. More specifically, a central unit 26is programmed to implement a processing method according to theinvention. Display or viewing means 27 make it possible, afterprocessing, to show the positioning of or the spatial distribution offluorophores in the medium 20 examined.

Other detection techniques can be used, for example an ultra-fastintensified camera of the “gated camera” type; in this case, the camerais opened in a time gate, with a width for example around 200 ps, thenthis gate is shifted, for example from 25 ps to 25 ps.

FIG. 1B is an example of another experimental system 2 using, as adetector 32, a fast camera. A beam 30 for excitation of the fluorescenceof a medium 20, containing one or more fluorophores 22, is emitted by aradiation source (not shown in the figure), which can be of the sametype as that shown above in association with FIG. 1A. A photodetector 36makes it possible to control means 40 forming a delay line. Reference 24designates, as in FIG. 1A, electronic data processing means of themicrocomputer type, programmed to store and process the data of thecamera 32. A central unit for these means 24 is programmed to implementa processing method according to the invention. Again, display orviewing means make it possible, after processing, to show thepositioning or the spatial distribution of fluorophores in the medium 20examined.

It is also possible to work with pulses in the femtosecond domain, onthe condition that there is an adequate radiation source, i.e. a lasersource 8 of which each pulse has a time width also in the femtoseconddomain.

A simple preliminary measurement makes it possible to measure theresponse (the abbreviation IRF for “Instrument Response Function” willhereinafter be used) of the device: the two fibres 10, 12 separated by apredetermined distance are aligned, and a pulse is transmitted from thefirst fibre 10 to the second fibre 12. The signal obtained at the outputof the second fibre 12 provides the IRF response. The width a mid-heightof this signal, in consideration of the distances used, can be on theorder of some dozens of ps, around 80 ps in the example provided.

A phantom can be used, containing distilled water in which a China ink(of Dalbe, France) is added as an absorbent medium, and “intralipid” (ofFresnius, France) is added as a diffusing medium. The relativeconcentrations are adjusted to the level of the parameters of thebiological tissues. The phantom is contained in a plastic cylinder witha diameter of 11 cm and a height of 10 cm. A volume of 1 μl offluorophore Cy5 (of Amersham) at a concentration of around 10 μmol·L−1is placed at the end of a thin glass capillary tube 21 (see FIG. 1B)(length 3 cm and thickness 1 mm). The tube is inserted into the phantomthrough a hole. The optical properties of the phantom and the lifetimeof the fluorophore are measured by TCSPC-type techniques (single photoncounting) and are presented in table I below.

TABLE I μ_(a) μ_(s)′ τ 0.0534 cm⁻¹ 11.82 cm⁻¹ 1.02 ns

In the measurements, the fibres are added at around 2.5 cm to the medium20, which makes it possible to approach the geometry of an infinitemedium.

Scans performed using computer means 24 (with software such as Labview),which simultaneously control the fibre movement means and the TCSPC card(or the camera 32 of FIG. 1B) with a parallel communication port.

We will first discuss the example of an infinite medium with a singlefluorophore.

We will consider a single fluorophore M in the medium, at an (unknown)position identified by a vector r at the point OXYZ. The excitationsource (or rather the end of the fibre 10, on the fluorophore side) islocated at r_(s) and the detector (or rather the end of the fibre 12, onthe fluorophore side) in position r_(d). The pulse transmission time isdesignated by t₀.

The source-fluorophore distance is denoted |r_(s)−r|=r_(sr) and thefluorophore-detector distance is denoted |r−r_(d)|=r_(rd).

In the time domain, the flow of excitation photons at the time t″ isdenoted φ_(x)(r_(sr), t″−t₀). The index x corresponds to the excitationwavelength λ_(x). The fluorophore 22 absorbs the excitation light andre-emits the fluorescence light at time t′, at the wavelength λ_(m), andwith a time decay τ. The expression of the photon flow emitted by thefluorophore located at r at the time t′ is the convolution of thepropagation function φ_(x) and the time decay of the fluorescence:

$\begin{matrix}{{{S_{f}\left( {r_{sr},t^{\prime}} \right)} = {\alpha {\int_{0}^{t^{\prime}}{{\varphi_{x}\left( {r_{rs},{t^{''} - t_{0}}} \right)}\frac{1}{\tau}{\exp \left( \frac{t^{\prime} - t}{\tau} \right)}{t^{''}}}}}}\;} & (1)\end{matrix}$

Where α is scale factor that is dependent on the power of illumination,the quantum efficiency of the fluorophore.

The equation 1 gives the temporal expression of the intensity of theradiation emitted by the fluorophore. This radiation will then bedetected by the detector 4 (in this case also, it is the end of thefibre, on the side of the medium 20), in position r_(d), at the time t,after having transited from the fluorophore 22 to this same detector.The resulting flow of fluorescence photons is proportional to a doubleconvolution:

-   -   for the propagation from the source to the fluorophore: the        convolution of φ_(x) (at the excitation wavelength) with the        fluorescence decay time, with this first convolution leading to        the above expression S_(f) (equation (1)),    -   and, then, that of S_(f) (given by the equation (1)) with φ_(m)        (at the emission wavelength), for the propagation of the        fluorophore to the detector.

It is therefore possible to write the detected flow of fluorescencephotons:

$\begin{matrix}\begin{matrix}{{\varphi_{fluo}\left( {r_{sr},R_{rd},t} \right)} = {\alpha {\int_{0}^{t}{{S_{f}\left( {r_{sr},t^{\prime},t_{0}} \right)}{\varphi_{m}\left( {{rd},{t - t^{\prime}}} \right)}\ {t^{\prime}}}}}} \\{= {\alpha {\int_{0}^{t}{\int_{0}^{t^{\prime}}{{\varphi_{x}\left( {r_{sr},t^{\prime},t_{0}} \right)}\frac{1}{\tau}{\exp \left( {- \frac{t^{\prime} - t^{''}}{\tau}} \right)}}}}}} \\{{{\varphi_{m}\left( {{rd},{t - t^{\prime}}} \right)}\ {t^{\prime}}{t^{''}}}}\end{matrix} & (2)\end{matrix}$

To have a simplified solution, it is possible to use the solution for aninfinite medium, with the approximation that the optical coefficientsare the same in excitation and in emission. The expression obtained isthe following:

$\begin{matrix}{{\varphi_{fluo}\left( {r_{sr},r_{r\; d},t} \right)} = {\alpha {\int_{0}^{t}\ {{t_{e}}\frac{r_{sr} + r_{r\; d}}{r_{sr}r_{r\; d}}\frac{1}{\left\lbrack {4\pi \; {c\left( {t - t_{e}} \right)}} \right\rbrack^{3/2}}{\exp \left\lbrack {{- \mu_{a}}{c\left( {t - t_{e}} \right)}} \right\rbrack}{\exp\left\lbrack {- \frac{\left( {r_{sr} + r_{r\; d}} \right)^{2}}{4{{cD}\left( {t - t_{e}} \right)}}} \right\rbrack}\frac{\exp \left( {{- t_{e}}/\tau} \right)}{\tau}}}}} & (3)\end{matrix}$

Where μ_(a) is the absorption coefficient and D is the diffusioncoefficient. For a plurality of fluorophores, we use, as indicatedbelow, an integral on the volume, and we assume a low-absorbing mediumsince this model does not take into account a possible reabsorption anda possible diffusion.

This theoretical function Φ_(fluo) corresponds to the curve, obtained byexperimental measurements, of FIG. 2B. A data processing operation, forexample a calculation of certain parameters, can be performed by takingdata in an entire time interval, from the start of the rising portion ofthe curve to the end of the falling portion, or at least by takingvalues in an interval on each side of the maximum of the curve, forexample a time interval of which the threshold values are thosecorresponding substantially to at least x % of the maximum intensity ofthe curve, with x for example being capable of being equal to 1 or 5 or10. In particular, it is possible on the basis of such data to calculatethe moments of any order 0, 1, . . . n (n>1).

It is possible in particular to extract from this function, thereforeequation 3, the mean time (or the first moment). For a distributionfunction g(t), the mean time is given by:

$\begin{matrix}{< t>=\frac{\int_{- \infty}^{\infty}{t\; {g(t)}\ {t}}}{\int_{- \infty}^{\infty}{{g(t)}\ {t}}}} & (4)\end{matrix}$

As shown, this first moment, like the moment of 0 order, can becalculated by taking into account the entirety, or almost, of the signaldetected, or the fluorescence function, in a large measurement window,and not only in the rising portion of the fluorescence curves.

To develop this formula, let us consider, rather than the temporalexpression of the flow, its frequency expression:

$\begin{matrix}{{\Phi_{fluo}\left( {r_{sr},r_{r\; d},\omega} \right)} = {\alpha^{\prime}\frac{\exp \left\lbrack {\; {k\left( {r_{sr} + r_{r\; d}} \right)}} \right\rbrack}{r_{sr} + r_{r\; d}}\frac{1}{1 - {\; \omega \; \tau}}}} & (5)\end{matrix}$

Where α′ is a constant independent of the frequency andk²=−μ_(a)/D+iω/(cD). Then, by using the following formula:

$\begin{matrix}\left. {< t>={\frac{\partial\Phi}{\partial\omega}}} \middle| {}_{\omega = 0}{\times \frac{1}{\left. {\Phi (\omega)} \right|_{\omega = 0}}} \right. & (6)\end{matrix}$

the analytical expression of the theoretical mean time can be found:

$\begin{matrix}{{< t >_{theo}} = {\frac{r_{sr} + r_{r\; d}}{2c\sqrt{\mu_{a}D}} + \tau}} & (7)\end{matrix}$

In this expression, the lifetime τ of the fluorophore appears. Theunknown position r is contained in the expressions of r_(sr) and r_(rd).

The processing of experimental data will now be described.

First, the calculation of the mean time on the experimental data can besensitive to the disturbances due to the excitation light or to thefluorescence of the medium surrounding the fluorophore, which occur whenthe fluorescence signal is low. In particular, the excitation light thatarrives first can reduce the mean time of the signal. To correct thisdisturbance, the signal can be measured, but without fluorophore.

This measurement without fluorophore is designated by mesure_(IL) and iscomposed:

-   -   partially by the excitation light that passes through the        filters,    -   and partially (long portion of the signal) by the fluorescence        of the surrounding medium, or the phantom without fluorophore.

This measurement can be subtracted from the data measured withfluorophore (mesure_(fluo)), if it is desirable to correct the residuallight data and obtain the real signal, designated by signal_(fluo) (toindicate the difference with a direct measurement, see equation 8). Thesubtraction also makes it possible to suppress the background noise. Bytaking into account the expressions of the detection times (respectivelyT_(fluo) for mesure_(fluo) and T_(IL) for mesure_(IL)), the fluorescencesignal can be expressed as follows:

$\begin{matrix}{{signal}_{fluo} = {{mesure}_{fluo} - {\frac{T_{fluo}}{T_{IL}}{mesure}_{IL}}}} & (8)\end{matrix}$

FIG. 3 gives the signal with fluorophore (curve I) and withoutfluorophore (curve II) for an inclusion at a depth z=0.74 cm. The endsof the fibres turned toward the medium 20 are separated by 0.2 cm.

For the calculation of the first moment, the integration thresholdvalues are defined by times defined by 1% of the time corresponding tothe maximum amplitude of the TPSF, for the upper and lower limit, so asto remove the background noise in accordance with the study of A.Liebert et al. “Evaluation of optical properties of highly scatteringmedia by moments of distributions of times of flight of photons” Appliedoptics, vol. 42, No. 28, 5785-5792 (2003).

More generally, the invention makes it possible to measure a signal in alarge time window, extending on each side of the time corresponding tothe maximum of the intensity signal as a function of time. The time dataon each side of this maximum can be used to calculate the variousmoments to be calculated.

To compare the mean time of the signal <t>_(signal) with the theoreticalmean time [equation 7], the IRF (function already defined above) can betaken into account. The signal is the convolution of the IRF with thetheoretical response or the specific response of the diffusing medium.The properties of the first moment show that the experimental mean timecan be written as the sum of the theoretical mean time <t>_(theo) andthe mean time of the IRF <t>_(IRF):

<t> _(signal) =<t> _(theo) +<t> _(IRF)  (9)

The reverse problem, i.e. determining the position of the inclusion as afunction of the measurements, can be solved for measurements above theinclusion (see FIG. 4, which shows the geometry of the ends of thefibres 10, 12 and the position of the fluorophore 22). As theinter-fibre distance is low (d=0.2 cm), it can be assumed thatr_(sr)+r_(rd)˜2z, where z represents the depth of the inclusion underthe fibres. It is therefore possible to rewrite the equation 9:

$\begin{matrix}\begin{matrix}{{< t >_{signal}} = {{\frac{r_{sr} + r_{r\; d}}{2c\sqrt{\mu_{a}D}} + \tau +} < t >_{IRF}}} \\{= {{\frac{z}{c\sqrt{\mu_{a}D}} + \tau +} < t >_{IRF}}}\end{matrix} & (10)\end{matrix}$

This expression can be reversed to obtain z when the optical parameters,the lifetime and the mean time of the IRF are known:

z=c√{square root over (μ_(a) D)}(<t> _(signal) −τ−<t> _(IRF))  (11)

However, this method can be used only if the ends of the fibres 10, 12are placed exactly above the inclusion so as to satisfy the assumptionr_(sr)+r_(rd)˜2z and to determine the lifetime and the mean time of theIRF.

To avoid this initial knowledge of the inclusion position, the lifetimeand the mean time of the IRF, a more general method has been developedto determine a completely unknown position.

A scanning is performed above the inclusion for N relativefluorophore-fibre positions. The relative position of two fibres,therefore of one fibre with respect to the other, preferably remainsconstant. The mean time, <t>_(signal,i), iε[1,N], is calculated for eachposition of the scanning. Then, another mean time, preferably thelowest: min(<t>_(signal,i)) is selected and subtracted from each meantime. This choice is justified by the fact that this lowest timecorresponds to the measurement position closest to the inclusion withthe best signal-to-noise ratio. Then, Δ<t>_(signal,i) is considered tobe the new variable:

$\begin{matrix}{\left. {{{\Delta < t >_{{signal},i}} = {< t >_{{signal},i}{- {\min \left( {< t >_{{signal},i}} \right)}}}},{i \in \left\lbrack {1,N} \right\rbrack}} \right) = {\frac{r_{{sr},i} + r_{{r\; d},i}}{2c\sqrt{\mu_{a}D}} - \frac{r_{{sr},\min} + r_{{r\; d},\min}}{2c\sqrt{\mu_{a}D}}}} & (12)\end{matrix}$

This new variable is independent of the lifetime and the mean time ofthe IRF due to the difference performed. It is instead dependent on theposition or the spatial distribution of the fluorophore. In the case ofa single fluorophore, this position of the inclusion, of coordinates(x,y,z), is then defined as the one enabling the error function χ² to beminimised.

$\begin{matrix}{{\chi^{2}\left( {x,y,z} \right)} = {\sum\limits_{i}\; \left( {\Delta < t >_{{signal},i}{- \Delta} < t >_{{theo},i}} \right)^{2}}} & (13)\end{matrix}$

The input variables are the values of Δ<t>_(signal,i) extracted from themeasurements. They are compared to the theoretical formula whereΔ<t>_(theo,i) is given by:

Δ<t> _(theo,i) =<t> _(theo,i)−min(<t> _(theo,j)), iε[1,N])  (14)

Where j is the index that identifies the experimental signal that hasthe shortest mean time. The adjustment procedure is, for example, basedon a simplex method such as that described in the article of J. C.Lagarias et al. “Convergence properties of the Nelder-Mead simplexmethod in low dimensions”, SIAM Journal on Optimization, vol. 9(1), p.112-147, 1998.

Experimental results will be presented, which make it possible tovalidate the model above. Then, we will present measurements that can,with the method according to the invention, solve the reverse problem,i.e. determine the position of the inclusion.

Time-resolved signals for an interfibre distance of 0.2 cm and forinclusions at different depths under the fibres (z=0.24; 0.34; 0.44;0.54; 0.64; 0.74 cm) are presented in a semi-logarithmic scale in FIG.5.

The amplitudes are shown in this figure in an arbitrary scale. In thisrepresentation, the exponential decay of the fluorescence of Cy5 appearsto tend toward a straight line. The more the depth z increases, the morethe time position of the maximum is shifted toward the long times, dueto the longer path of the photons.

The theoretical expression of the mean time was first validated. Themean time of the IRF was subtracted from the mean time measured so as toperform a comparison with the theoretical formula of equation 9.

FIG. 6 shows the corrected experimental mean time of the instrumentresponse (=<t>_(signal)−<t>_(IRF)) as a function of the depth z of theinclusion. In accordance with the theoretical expression (equation 10),the mean time is a linear function of the inclusion position. The slopemeasured experimentally is 1.14, equal to the theoretical slope(c√{square root over (μ_(a)D)})⁻¹ calculated on the basis of theequation (10) with the optical coefficients of the medium.

The ordinate at zero, 0.96 ns, is relatively close to the measuredlifetime (τ=1.02 ns).

Then, the fluorescence model was tested to determine whether itcorresponds to the measurements. The solution calculated (equation 3with the parameters of table I) is convoluted with the IRF and comparedwith the measurements. The curves are normalised at the same surface asthe experimental curve on an arbitrary time interval (1% of the maximumon the right and left sides).

FIG. 7 shows the experimental results for a fluorophore at the depthz=0.74 cm and two simulations for two different lifetimes (0.96 ns:solid line curve, 1.02 ns: dotted line curve). A good conformity isfound between the simulations and the experimental results.

Table II below presents the results of the reversal formula for τ=0.96ns and τ=1.02 ns. A good conformity is noted between the calculated andreal values.

TABLE II ^(z)exp (cm) 0.24 0.34 0.44 0.54 0.64 0.74 z calculated (τ =0.96 ns) 0.24 0.33 0.45 0.54 0.64 0.74 z calculated (τ = 1.02 ns) 0.190.28 0.40 0.49 0.59 0.69

The resolution of the reverse problem will be illustrated for variousinclusion depths using measurement grids.

The sampling is performed with a measurement grid comprising 5×5detectors arranged at a pitch of 0.2 cm. The grid is positioned abovethe inclusion. FIG. 8 shows the detector grid. The emission fibre 10 is−0.2 cm (according to axis x) from the detection fibre 13. The inclusionis centred at (0, 0) and placed at various depths. The centre (0, 0)corresponds to the collection fibre 12.

The adjustment procedure is applied to the data obtained from all of thedetection points. For the first depth (0.24 cm), for example, theexperimental value of the experimental differential mean time(Δ<t>_(signal)) and the differential mean time calculated by adjustmentare shown in FIG. 9. FIG. 10 shows the mean time as a function ofr_(sr)+r_(rd) (photon path), for an inclusion positioned at z=0.24 cm.Table III shows the results of the adjustment for each inclusion depthand the slopes corresponding to the plotting of the mean time as afunction of r_(sr)+r_(rd). These slopes can be compared to thetheoretical slope

$\begin{matrix}{\frac{1}{2c\sqrt{\mu_{a}D}},} & \left\lbrack {{Eq}.\mspace{14mu} (7)} \right\rbrack\end{matrix}$

calculated using the values of table I and equal to 0.572. Thetheoretical slope is therefore found, which is a confirmation of thevalidity of the model used. The device is that of FIG. 1A, but thefibres are separated by 2 mm and a scanning with a pitch of 2 mm isperformed above the inclusion (the phantom has a volume of around 1mm3). The position of the inclusion is identified with greater precisionthan the mm (200 μm for the set of measurements performed).

TABLE III z real (cm) x (cm) y (cm) z (cm) Slope 0.24 −0.01 −0.01 0.240.572 0.34 −0.02 0.01 0.33 0.577 0.44 −0.02 0.02 0.42 0.578 0.54 −0.020.04 0.53 0.577

Scans were performed with grids not centred on the inclusion.

The inclusion, initially positioned at z=0.3 cm under the fibres(initial position=(0; 0; 0.34)) was moved along the axis x toward twoother positions: (−0.2, 0, 0.34) then (−0.4, 0, 0.34). The zero positionis indicated only for comparison. As shown by table IV, which gives thepositions obtained by adjustment, by comparison with the real positions,both the location (x, y) and the depth are correctly determined by theadjustment procedure for the three locations.

TABLE IV Real (0, 0, 0.3) (−0.2, 0, 0.3) (−0.4, 0, 0.3) positionPosition by (−0.00, 0.00, (−0.20, 0.00, (−0.41, 0.00, adjustment 0.32)0.32) 0.31)

These tests show that the method does not require a priori knowledge ofthe inclusion position (x, y) and remains valid if the grid is shiftedwith respect to the inclusion.

As explained above in the case of a single fluorophore, the inventioncan also be applied to the case of a plurality of fluorophores, as willbe shown below. In this second case, it is attempted to find the spatialdistribution of the fluorophores in the medium 20. This spatialdistribution is the quantity to be reconstructed in the context of thereverse problem. The same type of experimental device as that used forthe single fluorophore, for example that of FIG. 1A or 1B, can be usedin the case of a plurality of fluorophores.

To this end we will consider, as in the first case, the theoreticalcontext of the approximation of the diffusion, in order to model thepropagation of the light in the diffusing medium. The equation of thediffusion that gives the density of photons Φ for a homogeneous mediumhas the form:

$\begin{matrix}{{{\frac{1}{c_{n}}\frac{\partial{\varphi \left( {r,t} \right)}}{\partial t}} - {D \cdot {\nabla^{2}{\varphi \left( {r,t} \right)}}} + {\mu_{a}{\varphi \left( {r,t} \right)}}} = {S\left( {r,t} \right)}} & (15)\end{matrix}$

where:

-   -   c_(n) is the propagation speed of the light in the medium. If        the refraction index of the medium is noted, then c_(n)=c/n with        c being the propagation speed of the light in the vacuum;    -   D=1/[3(μ_(a)+μ′_(s))] is the diffusion coefficient, where μ_(a)        and μ′_(s) are the reduced absorption and diffusion coefficients        of the medium.    -   S is the source term.

In the frequency domain, the solution is written:

$\begin{matrix}\begin{matrix}{{\Phi \left( {r,\omega} \right)} = {\frac{Q_{0}}{4\pi \; D}\frac{\exp \left( {\; {kr}} \right)}{r}}} \\{= {\frac{Q_{0}}{4\pi \; D}{G\left( {r,\omega} \right)}}}\end{matrix} & (16)\end{matrix}$

where Q_(o) is a factor dependent on the power of the source,

${G\left( {r,\omega} \right)} = \frac{\exp \left( {\; {kr}} \right)}{r}$

is the Green's function of the system and k is defined by the followingrelation:

$\begin{matrix}{k^{2} = {{- \frac{\mu_{a}}{D}} - \frac{\; \omega}{c\; D}}} & (17)\end{matrix}$

Other models can also be used (radiative transfer model, random stepmodel, or digital simulations of the Monte Carlo type, for example).This means that it is possible to calculate a Green's function incontexts other than the approximation of the diffusion.

Equations 15 and 16 above are the counterpart of the expression (1)given above in the case of a single fluorophore.

Let us consider a homogeneous diffusing medium that containsfluorophores.

A source pulse is transmitted in r_(s) at t₀, and a detector is placedin r_(d).

We note φ_(x)(|r−r_(s)|, t″) the photon density, which reaches the pointr at t″. The index “x” indicates that the wavelength is that of theexcitation source λ_(x).

No hypothesis is necessary on the form of Φ_(x). It can be noted that,if the medium is infinite, Φ_(x) will have the form given by thesolution (16) in an infinite medium.

A fluorophore placed in r will absorb this light Φ_(x) and emitfluorescence light at t′, with a fluorescence lifetime τ (also calledfluorescence decay) and an efficacy η. The photon density in r at t′ isthe convolution of the propagation function Φ_(x) and the fluorescencedecay:

$\begin{matrix}{{\delta \; {S_{f}\left( {{{r - r_{s}}},t^{\prime}} \right)}} = {\int_{0}^{t^{\prime}}{{\varphi_{x}\begin{pmatrix}{{\ {r - r_{s}}},} \\{t^{''} - t_{0}}\end{pmatrix}}\frac{{\eta (r)}{C(r)}}{\tau (r)}{\exp \left( {- \frac{t^{\prime} - t^{''}}{\tau (r)}} \right)}{t^{''}}}}} & (18)\end{matrix}$

We have denoted it as δS_(f) because this term then becomes a sourceterm at the emission wavelength λ_(m), which will be propagated anddetected in r_(d) at t. C(r) is proportional to the concentration offluorophores at point r. Hence, the expression of the final photondensity δφ_(fluo), which is the convolution of the previous term and thepropagation function Φm between the inclusion and the detector:

$\begin{matrix}\begin{matrix}{{{\delta\varphi}_{fluo}\left( {{{r - r_{s}}},{{r - r_{d}}},t} \right)} = {\int_{0}^{t}{\delta \; {S_{f}\left( {{{r - r_{s}}},t^{\prime}} \right)}\varphi_{m}}}} \\{{\left( {{{r_{d} - r}},{t - t^{\prime}}} \right){t^{\prime}}}} \\{= {\int_{0}^{t}{\int_{0}^{t^{\prime}}{\varphi_{x}\left( {{{r - r_{s}}},{t^{''} - t_{0}}} \right)}}}} \\{{\frac{{\eta (r)}{C(r)}}{\tau (r)}{\exp \left( {- \frac{t^{\prime} - t^{''}}{\tau (r)}} \right)}\varphi_{m}}} \\{{\left( {{{r_{d} - r}},{t - t^{\prime}}} \right){t^{\prime}}{t^{''}}}}\end{matrix} & (19)\end{matrix}$

It is possible to adopt the following notations to simplify the writing:

r _(sr) =|r−r _(s) |et, r _(rd) =|r−r _(d)|  (20)

If there is a plurality of fluorophores in the medium, the integrationis performed over the entire volume to obtain the final expression:

φ_(fluo)(r _(sr) ,r _(rd) ,t)=∫_(V) drδφ _(fl)(r _(sr) ,r _(rd),t)  (21)

For this volume integration, any reabsorptions and diffusions by theother fluorophores that are not taken into account in this model areoverlooked.

If the Fourier transform is performed on the previous expression, weobtain the expression of the fluorescence photon densityΦ_(f)(r_(s),r_(d),ω) in the frequency domain.

This expression has the advantage of being a bit simpler and moresuitable for the subsequent calculations:

$\begin{matrix}{{\Phi_{f}\left( {r_{sr},r_{r\; d},\omega} \right)} = {\int{\int{\int_{v}{{\Phi_{x}\left( {r_{sr},\omega} \right)}\frac{\eta (r){C(r)}}{1 + {\; \omega \; {\tau (r)}}}{\Phi_{m}\left( {r_{r\; d},\omega} \right)}{r^{3}}}}}}} & (22)\end{matrix}$

The fluorescence time curves are measured for a set of relativepositions on the one hand of the source-detector assembly and on theother hand of the medium 20. A data processing operation, for example acalculation of certain parameters, can be performed by taking data in anentire time interval, from the start of the rising portion of the curveto the end of the falling portion, or at least by taking values in aninterval on each side of the maximum of the curve, for example a timeinterval of which the threshold values are those correspondingsubstantially to at least x % of the maximum intensity of the curve,with x for example being capable of being equal to 1 or 5 or 10. Inparticular, it is possible on the basis of such data to calculate themoments of any order 0, 1, . . . n (n>1).

If we consider an infinite medium, at each point, the signal measured isproportional to the fluorescence photon density. The photon density canbe assimilated to the signal by assigning it a proportionalitycoefficient, which is dependent only on instrumental factors (excitationpower, detector gain, filter attenuation, etc.). The photon density isexpressed as a function of this instrumental factor, diffusioncoefficients D and Green's functions G, which are known (at bothwavelengths, which are always indicated by the indices x and m):

$\begin{matrix}{{\Phi_{f}\left( {r_{sr},r_{r\; d},\omega} \right)} = {\alpha \; {\int{\int{\int_{V}{\frac{1}{D_{x}D_{m}}{G_{x}\left( {r_{sr},\omega} \right)}\frac{\eta (r){C(r)}}{1 + {\; \omega \; {\tau (r)}}}{G_{m}\left( {r_{r\; d},\omega} \right)}{r^{3}}}}}}}} & (23)\end{matrix}$

This equation can be discretised by changing to a sum over the voxels,where k represents the size of the discretisation pitch:

$\begin{matrix}{{\Phi_{f}\; \left( {r_{sr},r_{r\; d},\omega} \right)} = {\alpha \; {\sum\limits_{v = {voxels}}{\frac{1}{D_{x}D_{m}}{G_{x}\left( {r_{{sr}_{v}},\omega} \right)}\; \frac{\eta \left( r_{v} \right){C\left( r_{v} \right)}}{1 + {\; \omega \; {\tau \left( r_{v} \right)}}}{G_{m}\left( {r_{r_{v}d},\omega} \right)}h^{3}}}}} & (24)\end{matrix}$

It is approximated that the optical coefficients are the same at bothwavelengths, and we therefore have: D=Dx=Dm.

Among all of the measurements, the source-detector pair is identified(denoted r_(s) _(m) −r_(d) _(m) ), for which the mean time of the TPSFis the shortest: this measurement is denoted φ^(min)(r_(s) _(m) ,r_(d)_(m) ). It is preferably chosen with respect to others, because it hasthe largest amplitude, and therefore the best signal-to-noise ratio fora constant power of the excitation light on the acquisitions.

The new functions considered are now defined by:

$\begin{matrix}{{\Phi^{N}\left( {r_{s},r_{d},\omega} \right)} = \frac{\Phi_{f}\left( {r_{s},r_{d},\omega} \right)}{\Phi^{m\; i\; n}\left( {r_{s_{m}},r_{d_{m}},\omega} \right)}} & (25)\end{matrix}$

For ω=0, the above formula gives the expression of the moment of order0, i.e. in fact the time integral of Φ_(fluo).

We can specify the expression of Φ^(min)(r_(s) _(m) , r_(d) _(m) ).

$\begin{matrix}{{\Phi^{m\; i\; n}\left( {r_{s_{m}},r_{d_{m}},\omega} \right)} = {\alpha \; {\sum\limits_{v = {voxels}}{\frac{1}{D^{2}}{G_{x}\left( {r_{s_{m}r_{v}},\omega} \right)}\frac{\eta \left( r_{v} \right){C\left( r_{v} \right)}}{1 + {\; \omega \; {\tau \left( r_{v} \right)}}}{G_{m}\left( {r_{r_{v}d},\omega} \right)}h^{3}}}}} & (26)\end{matrix}$

-   -   where the factor α remains the same factor as before, since the        experimental conditions have not changed.

We should note here the definition of the mean time m₁ for adistribution g(t):

$m_{1} = \frac{\int_{- \infty}^{\infty}{{{tg}(t)}{t}}}{\int_{- \infty}^{\infty}{{g(t)}{t}}}$

In the notations presented above, the denominator (the time integral ofg) of this expression constitutes m₀.

The mean time of the function Φ_(N) is written:

$\begin{matrix}{{m_{1}\left( \Phi^{N} \right)} = {m_{1}\left( \frac{\Phi_{f}}{\Phi^{m\; i\; n}} \right)}} \\{= {{m_{1}\left( \Phi_{f} \right)} - {m_{1}\left( \Phi^{m\; i\; n} \right)}}}\end{matrix}$

The expression of the mean time m₁(Φ_(f)) is as follows:

$\begin{matrix}{{m_{1}\left( \Phi_{f} \right)} = {\frac{1}{m_{0}\left( \Phi_{f} \right)}{\sum\limits_{v = {voxels}}\begin{Bmatrix}\begin{matrix}\left( {\frac{r_{{sr}_{v}} + r_{r_{v}d}}{2c\sqrt{\mu_{a}D}} + {\tau \left( r_{v} \right)}} \right) \\{\frac{1}{D^{2}}{G_{x}\left( {r_{{sr}_{v}},{\omega = 0}} \right)} \times}\end{matrix} \\{{G_{m}\left( {r_{r_{v}d},{\omega = 0}} \right)}\alpha \; h^{3}{\eta \left( r_{v} \right)}{C\left( r_{v} \right)}}\end{Bmatrix}}}} & (27)\end{matrix}$

-   -   and m₀(Φ_(f)) is written:

$\begin{matrix}{{m_{0}\left( \Phi_{f} \right)} = {\sum\limits_{v}{\frac{1}{D^{2}}{G_{x}\left( {r_{{sr}_{v}},{\omega = 0}} \right)}{G_{m}\left( {r_{r_{v}d},{\omega = 0}} \right)}\alpha \; h^{3}\; {\eta \left( r_{v} \right)}{C\left( r_{v} \right)}}}} & (28)\end{matrix}$

This amount is in fact known by the measurement: Φ_(f) is measured, andm₀(Φ_(f)) (as well as m₁(Φ_(f))) can be deduced from this function Φ_(f)obtained experimentally.

The term τ is dependent on the environment of the fluorophore. However,it can be assumed that this environment is the same for the differentfluorophores during a single measurement. Therefore, if the assumptionis made that τ is independent of r_(v), this term τ is removed from thesum, and it is possible to simplify the expression of m₁(Φ_(f)):

$\begin{matrix}{{m_{1}\left( \Phi_{f} \right)} = {{\frac{1}{m_{0}(\Phi)}{\sum\limits_{v}\begin{Bmatrix}\begin{matrix}\left( \frac{r_{{sr}_{v}} + r_{r_{v}d}}{2c\sqrt{\mu_{a}D}} \right) \\{\frac{1}{D^{2}}{G_{x}\left( {r_{{sr}_{v}},{\omega = 0}} \right)} \times}\end{matrix} \\{{G_{m}\left( {r_{r_{v}d},{\omega = 0}} \right)}\alpha \; h^{3}{\eta \left( r_{v} \right)}{C\left( r_{v} \right)}}\end{Bmatrix}}} + \tau}} & (29)\end{matrix}$

For m₁(Φ^(min)), the expression is the same by changing Φ by Φ_(min) andr_(s) by r_(sm) (respectively, rd by r_(dm)). Thus, in the calculationof the mean time of Φ^(N), the fluorescence lifetime disappears in thesubtraction, and the following is obtained:

$\begin{matrix}{{m_{1}\left( \Phi^{N} \right)} = {{\frac{1}{m_{0}\left( \Phi_{f} \right)}{\sum\limits_{v}\begin{Bmatrix}{\left( \frac{r_{{sr}_{v}} + r_{{r_{v}d}\;}}{2c\sqrt{\mu_{a}D}} \right)\frac{1}{D^{2}}{G_{x}\left( {r_{{sr}_{v}},{\omega = 0}} \right)}} \\{{G_{m}\left( {r_{r_{v}d},{\omega = 0}} \right)}\alpha \; h^{3}{\eta \left( r_{v} \right)}{C\left( r_{v} \right)}}\end{Bmatrix}}} - {\frac{1}{m_{0}\left( \Phi^{m\; i\; n} \right)}{\sum\limits_{v}\begin{Bmatrix}{\left( \frac{r_{s_{m}r_{v}} + r_{r_{v}d_{m}}}{2c\sqrt{\mu_{a}D}} \right)\frac{1}{D^{2}}{G_{x}\left( {r_{s_{m}r_{v}},{\omega = 0}} \right)}} \\{{G_{m}\left( {r_{r_{v}d_{m}},{\omega = 0}} \right)}\alpha \; h^{3}{\eta \left( r_{v} \right)}{C\left( r_{v} \right)}}\end{Bmatrix}}}}} & (30)\end{matrix}$

We can express this quantity linearly as a function ofαη(r_(v))C(r_(v)), the quantity to be reconstructed, by adding a weightfunction:

$\mspace{79mu} {{m_{1}\left( \Phi^{N} \right)} = {\sum\limits_{v}{P_{{({r_{s},r_{d}})},r_{v}}^{m_{0}m_{1}} \cdot \left\lbrack {\alpha \; {\eta \left( r_{v} \right)}{C\left( r_{v} \right)}} \right\rbrack}}}$  with:$P_{{({r_{s},r_{d}})},r_{v}}^{m_{0}m_{1}} = {{\frac{1}{m_{0}\left( \Phi_{f} \right)}\begin{Bmatrix}{\left( \frac{r_{{sr}_{v}} + r_{r_{v}d}}{2c\sqrt{\mu_{a}D}} \right)\frac{1}{D^{2}}{G_{x}\left( {r_{{sr}_{v}},{\omega = 0}} \right)}} \\{{G_{m}\left( {r_{r_{v}d},{\omega = 0}} \right)}h^{3}}\end{Bmatrix}} - {\frac{1}{m_{0}\left( \Phi^{m\; i\; n} \right)}\begin{Bmatrix}{\left( \frac{r_{s_{m}r_{v}} + r_{r_{v}d_{m}}}{{2c\sqrt{\mu_{a}D}}\;} \right)\frac{1}{D^{2}}{G_{x}\left( {r_{s_{m}r_{v}},{\omega = 0}} \right)}} \\{{G_{m}\left( {r_{r_{v}d_{m}},{\omega = 0}} \right)}h^{3}}\end{Bmatrix}}}$

Consequently, m₁(Φ^(N)) is measured, the ratios

$\frac{1}{m_{0}\left( \Phi_{f} \right)}\mspace{14mu} {and}\mspace{14mu} \frac{1}{m_{0}\left( \Phi^{m\; i\; n} \right)}$

are also measured and injected into the weight function P, which iscalculated, the system of linear equations is thus established,independently of the value of the fluorescence lifetime.

In other words, we return to the resolution of a system:

M=P×C

-   -   where M is a measurement column vector, P is a weight matrix        (which depends on the model chosen, therefore the function G and        quantities

$\frac{1}{m_{0}\left( \Phi_{f} \right)}\mspace{14mu} {and}\mspace{14mu} \frac{1}{m_{0}\left( \Phi^{m\; i\; n} \right)}$

and C an unknown column vector (vector proportional to the fluorophoreconcentrations). Once the system has been solved, the components ofvector C that can be represented 3 dimensionally are known.

The solution can therefore be represented with an operator on displaymeans such as means 27 of FIG. 1A or 1B.

The data processing means 24 make it possible to solve a system ofequations such as the system above, and are therefore programmed forthis purpose.

The resolution of the problem of the location or spatial distribution offluorophores can implement one or more of the various techniquesdescribed, for example, in A. C. Kak et al. “Principles of computerizedtomographic imaging”, IEEE, NY, 1987.

The data can also be acquired in frequency mode at various frequenciesso as to reconstruct the TPSFs by performing a Fourier transform.

The developments above show that it is possible to use, in the contextof the invention, a function of the frequency. This frequency functioncan be normalised, for example with respect to the frequency functionthat corresponds to the minimum mean time.

1. Method for processing fluorescence signals emitted, after excitationby radiation from a radiation source, by at least one fluorophore with alifetime τ in a surrounding medium, which signals are detected bydetection means, the method comprising: detecting, for each pair of afirst position of the radiation source and a second position of thedetection means, a fluorescence signal Φ_(fluo) emitted by said at leastone fluorophore in its surrounding medium via a detector; calculating,based on the detected fluorescence signals, values of a variable,independent of the lifetime τ using a computer; and determining aposition or a spatial distribution and/or a concentration of said atleast one fluorophore in said medium on the basis of the values of saidvariable.
 2. Method according to claim 1, wherein said variable isindependent of τ resulting from a frequency normalised function. 3.Method according to claim 2, wherein said function being frequencynormalised with respect to a specific fluorescence signal.
 4. Methodaccording to claim 1, wherein said variable is independent of τresulting from a difference between a first mean time calculated foreach fluorescence signal and a second mean time calculated for aspecific fluorescence signal.
 5. Method according to claim 3, whereinsaid specific fluorescence signal has a minimum mean time or a minimumcalculated mean time.
 6. Method according to claim 4, wherein saidspecific fluorescence signal has a minimum mean time or a minimumcalculated mean time.
 7. Method according to claim 1, wherein saidvariable is calculated from Mellin-Laplace transforms on the basis offluorescence signals or moments of orders greater than the normalisedfluorescence functions.
 8. Method according to claim 1, wherein theposition or the spatial distribution of said at least one fluorophore insaid medium is determined by a method of reversal technique using valuesof said variable.
 9. Method according to claim 8, wherein the positionor the spatial distribution is determined using a minimisation of anerror function using a simplex function.
 10. Method according to claim1, wherein the position or the spatial distribution of said at least onefluorophore in said medium is determined by implementing a system oflinear equations comprising:M=P×C, where M is a measurement column vector, P is a weight matrix andC is a distribution column vector.
 11. Method according to claim 1,further comprising calculating a first moment of a fluorescence curve ofthe fluorescence signals as a function of time.
 12. Method according toclaim 1, further comprising displaying a visual or graphicrepresentation of the position or the spatial distribution of the atleast one fluorophore.
 13. Method according to claim 1, wherein theradiation is of a femtosecond type.
 14. Method according to claim 1,wherein the fluorescence signals are detected by a TCSPC technique or bycamera.
 15. Method according to claim 1, further comprising measuringthe fluorescence signals by the surrounding medium, in absence of afluorophore; and correcting the fluorescence signals Φ_(fluo) emitted bythe fluorophore in the surrounding medium.
 16. Method according to claim1, wherein the first position of the radiation source is an end of anoptical fiber which transmits said radiation from said radiation sourceto said medium and to said fluorophore.
 17. Method according to claim 1,wherein the second position of the detection means is an end of anoptical fiber which collects light from at least said fluorophore andtransmits it to said detection means.
 18. Method according to claim 1,wherein the surrounding medium is human or animal tissue.
 19. Device forprocessing fluorescence signals emitted by at least one fluorophore,with a lifetime τ in a surrounding medium, comprising: a source ofradiation for excitation of said at least one fluorophore, means fordetecting a fluorescence signal emitted by said fluorophore, wherein thesource or the detection means are moveable relative to said at least onefluorophore; means for calculating values of a variable independent ofτ, on the basis of a plurality of fluorescence signals Φ_(fluo) emittedby the fluorophore into its surrounding medium, with each fluorescencesignal corresponding to a relative position of the fluorophore and thesource and the detection means to determine a position or a spatialdistribution and/or a concentration of said at least one fluorophore insaid medium based on the calculated values of said variable.
 20. Deviceaccording to claim 19, wherein said detection means comprising aTCSPC-type means or a camera-type means.
 21. Device according to claim19, further comprising means for visual or graphic representation of theposition or the spatial distribution of the at least one fluorophore.22. Device according to claim 19, wherein said variable is independentof τ resulting from a normalised frequency function.
 23. Deviceaccording to claim 22, wherein said function is frequency normalizedwith respect to a specific fluorescence signal.
 24. Device according toclaim 19, wherein said variable is independent of τ resulting from adifference between a mean time τ calculated for each fluorescence signaland a mean time calculated for a specific fluorescence signal. 25.Device according to claim 23, wherein said specific fluorescence signalhas a minimum mean time or a minimum calculated mean time.
 26. Deviceaccording to claim 24, wherein said specific fluorescence signal has aminimum mean time or a minimum calculated mean time.
 27. Deviceaccording to claim 19, wherein said variable is calculated fromMellin-Laplace transforms of the fluorescence signals or moments ofgreater orders of normalised fluorescence functions.
 28. Deviceaccording to claim 19, wherein said means for determining the positionor the spatial distribution of said at least one fluorophore in saidmedium implements a method of reversal technique using values of saidvariable.
 29. Device according to claim 19, wherein statisticalprocessing of the values of said variable being a minimisation of anerror function using a simplex method.
 30. Device according to claim 19,wherein the source further comprises a first optical fiber coupledthereto, wherein an excitation signal is transmitted from the source tothe fluorophore via an end of the fiber.
 31. Device according to claim19 wherein the detection means further comprises a second optical fibercoupled thereto, the second optical fiber configured to transmit thefluorescence signal to the detection means via an end of the fiber. 32.Device according to claim 30, wherein each fluorescence signalcorresponds to the relative position of the fluorophore and the end ofthe first optical fiber coupled to the source.
 33. Device according toclaim 31, wherein each fluorescence signal corresponds to the relativeposition of the fluorophore and the end of the second optical fiber. 34.Device according to claim 16, wherein the source and the detection meansare moveable relative to said at least one fluorophore.
 35. Device forprocessing fluorescence signals emitted by at least one fluorophore,with a lifetime τ in a surrounding medium, the device comprising: asource of radiation for excitation of said at least one fluorophore, thesource including a first optical fiber coupled thereto and having an endmoveable relative to the at least one fluorophore, wherein an excitationsignal is transmitted from the source to the fluorophore via the end ofthe fiber; a detector configured to detect a fluorescence signal emittedby said fluorophore in response to the excitation signal, the detectorincluding a second optical fiber having an end moveable relative to theat least one fluorophore, wherein the fluorescence signal is transmittedto the detector via the end of the second optical fiber; and a computerconfigured to calculate values of a variable independent of τ, on thebasis of a plurality of fluorescence signals Φ_(fluo) emitted by thefluorophore into its surrounding medium, with each signal correspondingto a relative position of the fluorophore and the movable end of saidfirst optical fiber and the movable end of said second optical fiber todetermine a position or a spatial distribution and/or a concentration ofsaid at least one fluorophore in said medium based on the calculatedvalues of said variable.
 36. Device for processing fluorescence signalsemitted by at least one fluorophore, with a lifetime τ in a surroundingmedium, the device comprising: a source of radiation for excitation ofsaid at least one fluorophore, the source including an optical fibercoupled thereto and having an end moveable relative to the at least onefluorophore, wherein the excitation signal is transmitted from thesource to the fluorophore via the end of the fiber; a detectorconfigured to detect a fluorescence signal emitted by said fluorophore,the detector being moveable relative to the at least one fluorophore;and a computer configured to calculate values of a variable independentof τ, on the basis of a plurality of fluorescence signals Φ_(fluo)emitted by the fluorophore into its surrounding medium, with each signalcorresponding to a relative position of the fluorophore and said end ofsaid optical fiber and the detector to determine a position or a spatialdistribution and/or a concentration of said at least one fluorophore insaid medium based on the calculated values of said variable.
 37. Devicefor processing fluorescence signals emitted by at least one fluorophore,with a lifetime τ in a surrounding medium, the device comprising: asource of radiation for excitation of said at least one fluorophore; adetector configured to detect a fluorescence signal emitted by saidfluorophore, the detector including an optical fiber having an endmoveable relative to the at least one fluorophore, wherein thefluorescence signal is transmitted to the detector via the end of saidoptical fiber; and a computer configured to calculate values of avariable independent of τ, on the basis of a plurality of fluorescencesignals Φ_(fluo) emitted by the fluorophore into its surrounding medium,with each signal corresponding to a relative position of the fluorophoreand said radiation source and the end of said optical fiber to determinea position or a spatial distribution and/or a concentration of said atleast one fluorophore in said medium based on the calculated values ofsaid variable.